The ultimate objective of this project is to understand mechanisms of chemical/drug induced liver injury. There is increasing evidence that leukocytes play a critical role in liver injury and the focus of this project is on defining the toxic mechanisms of the macrophage-derived cytokine, Tumor Necrosis Factor-alpha (TNFalpha). Experiments conducted in this laboratory have shown that TNFalpha exerts direct effects on hepatocytes which include ATP depletion, followed by cytotoxicity when the glutathione anti-oxidant defense system is impaired. ATP depletion is preventable by free radical scavengers and it is preceded by evidence of an oxidant-mediated stress. It is proposed to conduct experiments to further our understanding of TNFalpha's direct effects on hepatocytes and to explore the hypothesis that TNFalpha causes hypoxic injury in hepatocytes by inhibiting mitochondrial respiration, possibly due to alterations in calcium homeostasis. Leakage of oxygen radicals from the inhibited mitochondrial electron transport chain, coupled with increased cytosolic generation of oxygen radicals, may account for the oxidant-mediated stress. Furthermore, the direct effects of TNFalpha on hepatocytes may be potentiated by other macrophage-derived substances which are simultaneously present in the intact animal. Experiments will be conducted with primary cultures of rodent hepatocytes treated with recombinant murine TNFalpha. Receptor binding, uptake, and degradation of TNFalpha will be measured (Specific Aim 1) as well as the effects of TNFalpha on mitochondrial structure and function (Specific Aim 2) and cytosolic free calcium ion concentrations (Specific Aim 3). In some experiments, hepatocytes will be exposed to low oxygen tension in order to simulate the situation which occurs when TNFalpha altered endothelial cell function leads to ischemia (Specific Aim 4). In Specific Aim 5, TNFalpha's direct effects on hepatocytes will be compared with the effects of conditioned medium from endotoxin- or TNFalpha-treated Kupffer cells. This medium will contain TNFalpha as well as other cytokines and potentially synergistic injurious agents.